Ca2+ signaling dysfunction occurs in heart muscle under many conditions and is associated with diverse cardiac pathologies. This project seeks to characterize quantitatively Ca2+ signaling defects that occur in cardiac ventricular myocytes and to investigate the causes of the dysfunction. Confocal Ca2+ imaging of single ventricular myocytes will be carried out to measure Ca2+ sparks and the cellular Ca2+ signal at high temporal and spatial resolution. Intracellular Ca2+ will be rapidly changed by photorelease of caged Ca2+ while membrane potential is controlled with a whole cell patch clamp method. The planned experiments will reveal the links between the Ca2+ current, the Ca2+ sparks and other components of the Ca2+ signal. Preliminary experiments suggest a central hypothesis: a disruption of the machinery of excitation-contraction coupling may underlie the Ca2+ signaling defects that have been observed. Such remodeling may involve reorganization of the cytoskeletal proteins, altered positioning of L-type Ca2+ channel proteins and/or the ryanodine receptor Ca2+ release channels or re-organization of the transverse-tubules. To investigate the hypothesis, transgenic mice, rat models of disease and new cell culture methods will be used to examine the molecular causes of the Ca2+ signaling defects. Specific experiments will be carried out to characterize the role of cytoskeletal elements in the Ca2+ signaling defects. The planned work should therefore broaden our understanding of Ca2+ signaling in heart and clarify how it may become dysfunctional in disease. Additionally, the work should provide insight into the subcellular organization of the heart cell and illuminate the links between transverse tubules, cytoskeletal structures, and voltage- and ligand-gated channels.